Process for extracellular secretion of Brazzein

ABSTRACT

The present invention discloses a process for the secretion of brazzein in improved yield.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process for the extracellularsecretion of Brazzein from E. coli cells in enhanced yield and purity.

BACKGROUND OF THE INVENTION

Brazzein is a high-potency thermostable sweet protein, originallyisolated from the fruit of the West African climbing tree Pentadiplandrabrazzeana Baillon. (Ming and Hellekant, FEBS Lett. 355: 106-108, 1994).Brazzein is 2,500 times sweeter than sucrose in comparison to 2% sucroseaqueous solution and 500 times in comparison to 10% sucrose. Due to itsclose taste profile with sucrose compared to other protein based naturalsweetners such as thaumatin or monellin, its water solubility, stabilityat high temperatures and at low pH, brazzein may be conveniently used inbaking formulations and by industrial food manufacturers. This stabilityof Brazzein coupled with its sweet taste profile makes this a suitablealternative to currently available low calorie high intensity sweetenerssuch as sucralose, Aspartame, and Stevia. As a dietary protein, it issafe for consumption by diabetics. When blended with other sweetenerssuch as aspartame and stevia, Brazzein reduces their aftertaste andcomplements their flavour.

However, isolation of Brazzein from it natural source is expensive, andhence is not commercially feasible. Moreover, Pentadiplandra brazzeana,known for producing Brazzein which is found in the extracellular spaceof the pulp surrounding the seeds is geographically located in the WestAfrican regions of Cameroon and Gabon, isolating brazzein on acommercial scale from other regions globally becomes impractical.

In view of the many difficulties resulting from the process ofextraction of Brazzein from P. brazzeana, several synthetic approacheshave been proposed in the art to devise a mechanism for the synthesis ofBrazzein.

Structurally, Brazzein comprises 54 amino acid residues, correspondingto a molecular mass of 6.5 kDa, and has been classified based onvariations at the N-terminal sequences. Type I Brazzein is an unstableform composed of 54 amino acids having glutamine as the first aminoacid. This unstable form may be converted to Type II Brazzein form orthe Major form, wherein glutamine at the N-terminal position isconverted to pyroglutamate. In Type III Brazzein or Minor form,glutamine is absent at the first amino acid position, therefore spanningan amino acid sequence of 53 amino acids. (Assadi-Porter, et al., Arch.Biochem. Biophys. 376: 252-258, 2000).

Other than these naturally existing forms, researchers have developedvariants and multi variants of brazzein having excellent physicalstability at varying temperatures, and pH ranges and possessing anexcellent taste profile by mutating wild-type Brazzein throughsubstitution of amino acids at certain positions.

A disclosure in U.S. Pat. No. 8,592,181 relates to Brazzein variants andmulti variants having higher sweetness and a method for preparing thesaid multi-variant. A corresponding research article (Kwang-Hoon Kong etal Bull. Korean Chem. Soc. 2010, Vol. 31, No. 12) by the same inventorof LTS'181 has, to improve production levels of the recombinant solublebrazzein, established a new strategy using the pelB leader sequence ofpectate lyase B from Erwinia carotovora CE for Brazzein expression. Theprocess for Brazzein expression disclosed therein only resulted inperiplasmic localization of the protein. Post fermentation, downstreamprocessing techniques involved subjecting the induced BL21 Star (DE3)cells to centrifugation and osmotic shock. Major disadvantages ofperiplasmic expression and osmotic treatment to isolate the protein areits low expression yields, several centrifugation steps and a largeincrease in buffer volumes during the osmotic treatment, makingisolation processes tedious and results in yield loss.

Fariba Assadi-Porter et al., (Protein Expr Purif. 2008 April;58(2):263-8) have attempted at providing for efficient and rapid proteinexpression and purification of Brazzein in E. coli. However, the methoddescribed therein involves intracellular expression of the Brazzeinprotein fused to Small Ubiquitin-like Modifier protein (SUMO), followedby cleavage by SUMO protease. SUMO proteins are a family of smallproteins which are covalently attached to and detached from otherproteins in cells to modify their function. However, fusion of SUMO tothe Brazzein protein does not modify the localization of Brazzein to theextracellular region, thereby resulting in usage of expensive andinconvenient downstream processes to isolate and purify the functionalprotein.

In a research article by Fariba Assadi-Porter et al., (Arch BiochemBiophys. 2000, 15; 376(2):252-8) Brazzein in fusion with Staphylococcalnuclease protein with single engineered cyanogen bromide cleavage sitebetween Brazzein and Staphylococcal nuclease has been expressed. Thisfusion protein is expressed in insoluble inclusion bodies in E. coli,thus requiring extensive refolding of the followed by cleavage withcyanogen bromide to isolate functional Brazzein.

The above disclosures, relate either to periplasmic or intracellularlocalization of Brazzein in E. coli, however no attempts have been madein prior art to obtain extracellular Brazzein secretion when expressedin E. coli in order to increase expression yield and make downstreamprocessing convenient and economical during industrial scale Brazzeinproduction.

Extracellular release of proteins lodged in the periplasmic space isusually achieved by several strategies like employing leaky strains forprotein expression, cell membrane permeabilization, or co-expression ofrelease proteins. However, applications of these techniques incurexorbitant costs.

In view of the shortcomings of the methods known in the art, the presentinventors have with the object of minimizing disadvantages posed by lowexpression yields and downstream processing of Brazzein protein,provided a convenient process for extracellular secretion of Brazzeinprotein from E. coli cells in order to increase expression yields andease downstream processing techniques.

SUMMARY OF THE INVENTION

In a preferred aspect, the present invention provides a process for theextracellular secretion of recombinant Brazzein with improved yieldcomprising:

-   -   (a) ligating a plasmid with a nucleotide sequence encoding a        signal leader sequence conjugated to Brazzein to obtain a        recombinant DNA construct and inserting the said construct in        BL21(DE3) E. coli cells by transformation;    -   (b) culturing the transformed cells of step (a) carrying the        recombinant construct in a culture medium in the presence of        Dextrose and inducing protein expression with inducing agents        selected from IPTG or lactose for a period ranging from 4 hrs to        72 hrs to obtain secretion of brazzein in the culture medium,        and    -   (c) separating culture medium from cells after 4 to 72 hours        post induction and purifying Brazzein from the clarified medium.

Accordingly, the nucleotide sequence to be ligated in the recombinantvector is such that it encodes a signal leader sequence fused toBrazzein. The signal leader sequence is selected from the groupconsisting of pelB, ompA, Bla, PhoA, PhoS, MalE, LivK, LivJ, MglB, AraF,AmpC, RbsB, MerP, CpdB, Lpp, LamB, OmpC, PhoE, OmpF, TolC, BtuB and LutAsignal sequences.

In another aspect, the present invention provides a process forextracellular secretion of recombinant Brazzein comprising (a) insertingthe pelB-Brazzein nucleotide sequence in a pET vector, wherein the saidpelB-Brazzein sequence is amplified by employing primers selected fromsequences represented by Seq Id No. 3, Seq Id No. 4; (b) ligatingpelB-Brazzein in a pET vector to obtain a recombinant construct andinserting the said construct into BL21(DE3) E. coli cells bytransformation; (c) culturing the transformed cells of step (b) carryingthe recombinant construct in a culture medium in the presence ofDextrose and inducing protein expression with inducing agents selectedfrom IPTG or lactose, (d) separating culture medium from cells after 4to 72 hours post induction and purifying Brazzein from the clarifiedmedium.

In yet another aspect, Brazzein expressed extracellularly in the culturemedium is selected from the group consisting of the naturally existingwild-type functional type III Brazzein, a variant comprising asubstitution at the 28^(th) position of the type III brazzein fromAspartate to Alanine D28A or multi-variant of the type III.

In one aspect, the present invention provides a process forextracellular secretion of Brazzein in enhanced yield, wherein the yieldof brazzein ranging between 0.5 g/l to 5 g/l. The present process isperformed on a fermenter scale or at a laboratory stage in shaker flasksystem.

DETAILED DESCRIPTION OF DRAWINGS

FIG. 1 depicts a vector construct comprising a lac I Promoter, LacIgene, lac operator, T7 promoter, T7 terminator and pelB-Brazzein codingnucleotide sequence.

FIG. 2 depicts PCR amplification of pelB-Brazzein recombinant constructwherein Lane 1 shows amplification product corresponding topelB-Brazzein and Lane 2 shows 100 bp DNA ladder.

FIG. 3 depicts IPTG induced whole cell expression of Brazzein, depictedby 6.5 kiloDalton Brazzein protein. M represents Low weight molecularweight marker, Lane 1: un-induced whole cell extract, Lane 2: Whole cellextract 24 hours post-induction with 0.25 mM IPTG, Lane 3: Whole cellextract 48 hours post-induction with 0.25 mM IPTG. Lane 4: Whole cellextract 72 hours post-induction with 0.25 mM IPTG, Lane 5: Whole cellextract 24 hours post-induction with 0.50 mM IPTG, Lane 6: Whole cellextract 48 hours post-induction with 0.50 mM IPTG, Lane 7: Whole cellextract 72 hours post-induction with 0.50 mM IPTG.

FIG. 4 depicts IPTG induced extracellular secretion of Brazzein in LBmedium. M: Low weight molecular weight marker. Lane 1: Cell freesupernatant of uninduced cells, Lane 2: Cell free supernatant after 24hours induction with 0.25 mM IPTG, Lane 3: Cell free supernatant after48 hours induction with 0.25 mM IPTG, Lane 4: Cell free supernatantafter 72 hours induction with 0.25 mM IPTG, Lane 5: Cell freesupernatant after 24 hours induction with 0.50 mM IPTG, Lane 6: Cellfree supernatant after 48 hours induction with 0.50 mM IPTG, and Lane 7:Cell free supernatant after 72 hours induction with 0.50 mM IPTG;

FIG. 5 depicts Lactose induced extracellular secretion of Brazzein in LBmedium, M represents the Low weight molecular weight marker, Lane 1:Cell free supernatant of uninduced cells, Lane 2: Cell free supernatantafter 48 hours induction with 2.5 mM Lactose, Lane 3: Cell freesupernatant after 72 hours of induction with 2.5 mM Lactose, Lane 4:Cell free supernatant after 48 hours induction with 5.0 mM Lactose, andLane 5: Cell free supernatant after 72 hours induction with 5.0 mMLactose;

FIG. 6 depicts Lactose induced extra-cellular secretion of Brazzein inTB medium, M represents the Low weight molecular weight marker, Lane 1:Cell free supernatant of uninduced cells, Lane 2: Cell free supernatantafter 2 hours of induction with 5 mM Lactose, Lane 3: Cell freesupernatant after 18 hours of induction with 5 mM Lactose, Lane 4: Cellfree supernatant after 24 hours of induction with 5.0 mM Lactose, andLane 5: Cell free supernatant after 36 hours of induction with 5.0 mMLactose, Lane 6: Cell free supernatant after 48 hours of induction with5.0 mM Lactose;

FIG. 7 depicts Lactose induced extra-cellular secretion ofpelB-Brazzein(A28D) in TB media, M represents the Low weight molecularweight marker, Lane 1: Cell free supernatant of uninduced cells, Lane 2:Cell free supernatant after 4 hours of induction with 5 mM Lactose, Lane3: Cell free supernatant after 18 hours of induction with 5 mM Lactose,Lane 4: Cell free supernatant after 24 hours of induction with 5.0 mMLactose, and Lane 5: Cell free supernatant after 36 hours of inductionwith 5.0 mM Lactose, Lane 6: Cell free supernatant after 48 hours ofinduction with 5.0 mM Lactose. The position of mature Brazzein(A28D) isindicated by the arrow.

FIG. 8 depicts lactose induced extra-cellular secretion ofompA-Brazzein(A28D) in TB medium, M: Low weight molecular weight marker,Lane 1: Cell free supernatant of uninduced cells, Lane 2: Cell freesupernatant after 4 hours of induction with 5 mM Lactose, Lane 3: Cellfree supernatant after 18 hours of induction with 5 mM Lactose, Lane 4:Cell free supernatant after 24 hours induction with 5.0 mM Lactose, andLane 5: Cell free supernatant after 36 hours induction with 5.0 mMLactose, Lane 6: Cell free supernatant after 48 hours induction with 5.0mM Lactose.

FIG. 9 depicts Lactose induced extra-cellular secretion ofpelB-Brazzein(A28D) in a Fermentor, M represents the Low weightmolecular weight marker, Lane 1: Cell free supernatant of uninducedcells, Lane 2: Cell free supernatant after 2 hours of induction with 5mM Lactose, Lane 3: Cell free supernatant after 12 hours of inductionwith 5 mM Lactose, Lane 4: Cell free supernatant after 18 hours ofinduction with 5.0 mM Lactose, and Lane 5: Cell free supernatant after24 hours of induction with 5.0 mM Lactose. The position of matureBrazzein(A28D) is indicated by the arrow.

FIG. 10 depicts purification of Brazzein(A28D) from cell culturesupernatant after expression in a fermentor. Lane 1: clarified sampleafter ammonium sulfate precipitation; Lane 2: final purified sample.

FIG. 11 depicts thermal stability of secreted brazzein synthesized bythe present process, which was subjected to treatment at hightemperature. M depicts the Low weight molecular weight marker. Lane 1:Untreated control sample of the cell free supernatant from a 72 hourlactose induced culture. Lane 2: Same as in Lane 1, except that the cellfree supernatant was heated at 80° C. for 60 mins, centrifuged at 17,500g and the soluble supernatant was analysed. Presence of Brazzein inlanes 1 and 2 is indicated by an arrow.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail in connection with certainpreferred and optional embodiments, so that various aspects thereof maybe more fully understood and appreciated.

In a preferred embodiment, the present invention provides a process forthe extracellular secretion of recombinant brazzein with improved yieldcomprising:

-   -   (a) ligating a nucleotide sequence encoding a signal leader        sequence conjugated to Brazzein in a recombinant vector to        obtain a recombinant DNA construct and inserting the said        construct in BL21(DE3) E. coli cells by transformation;    -   (b) culturing the transformed cells of step (a) carrying the        recombinant construct in a culture medium in the presence of        Dextrose and inducing protein expression with inducing agents        selected from IPTG or lactose for a period ranging from 4 hrs to        72 hrs to obtain secretion of Brazzein in the culture medium,        and    -   (c) separating culture medium from cells and purifying Brazzein        from the clarified medium

Accordingly, nucleotide sequence encoding the signal leader sequenceconjugated to the Brazzein is amplified with primers. The signalsequence-Brazzein amplified gene is ligated in an expression vector toobtain a recombinant vector construct which is transformed in BL21(DE3)E. coli cells. The transformed cells of E. coli carrying the recombinantconstruct is cultivated in a culture medium in the presence of Dextroseand inducing protein expression with inducing agents selected from IPTGor lactose for a period ranging from 4 hrs to 72 hrs, to obtain theextracellular secretion of brazzein. The cells are separated from theculture medium post induction and the culture medium also termed as theextracellular fraction is subjected to heating at temperatures as highas 90° C. to obtain Brazzein having >90% purity and in increased yield.As a result of heating, most of the endogenous E. coli proteins presentin the cell free supernatant are precipitated, leaving >90% pureBrazzein in solution.

In an embodiment, the signal leader sequence to be conjugated toBrazzein is selected from the group consisting of the pelB s ompA, Bla,PhoA, PhoS, MalE, LivK, LivJ, MglB, AraF, AmpC, RbsB, MerP, CpdB, Lpp,LamB, OmpC, PhoE, OmpF, TolC, BtuB and LutA signal sequences.

In another embodiment, the present invention provides induction ofsignal leader-Brazzein expression in E. coli cells by addition oflactose ranging from 2.5 mM to 5 mM in the culture medium.

In yet another embodiment, the present invention provides induction ofsignal leader-Brazzein expression in E. coli cells by IPTG additionranging from 0.25 mM to 0.5 mM in the culture medium.

Accordingly, extra-cellular secretion of brazzein at varyingconcentration of IPTG i.e. 0.25 mM and 0.5 mM at durations ranging from24 hrs to 72 hrs is depicted in FIG. 4. Extra-cellular secretion ofbrazzein at varying concentration of lactose, i.e. 2.5 mM and 5 mM atdurations ranging from 2 hrs to 72 hrs is depicted in FIG. 5 and FIG. 6.Post addition of the inducing agents in the culture medium, growth ofcells is performed at temperatures ranging from temperatures between 25°C. to 37° C. and subjecting the cells to shaking conditions.

In another preferred embodiment, the present invention provides aprocess for extracellular synthesis of recombinant Brazzein withimproved yield comprising:

-   -   a) ligating a pET vector with a nucleotide sequence encoding a        pelB signal leader sequence conjugated to Brazzein to obtain a        recombinant DNA construct and inserting the said construct in        BL21(DE3) E. coli cells by transformation;    -   b) culturing the transformed cells of step (a) carrying the        recombinant construct in a culture medium in the presence of        Dextrose and inducing protein expression with inducing agents        selected from IPTG or lactose for a period ranging from 2 hrs to        72 hrs, and    -   c) separating culture medium from cells 4-72 hours post        induction and purifying Brazzein from the clarified medium.

Accordingly, the process for extracellular synthesis of recombinantBrazzein conjugated to the pelB signal leader sequence comprises (a)inserting the pelB-Brazzein coding sequence in a vector, wherein thesaid coding sequence is amplified by employing primers selected fromsequences represented by Seq Id No. 3 and Seq Id No. 4; (b) ligatingpelB-Brazzein coding sequence in a pET vector to obtain a recombinantconstruct and inserting the said construct in BL21(DE3) E. coli cells bytransformation; (c) culturing the transformed cells of step (b) carryingthe recombinant construct in a culture medium in the presence ofDextrose and inducing protein expression with inducing agents selectedfrom IPTG or lactose, (d) separating culture medium from cells postinduction and purifying Brazzein from the clarified medium.

Accordingly, the pET recombinant vector employed to carry thepelB-Brazzein nucleotide sequence is depicted in FIG. 1 of the presentinvention.

The molecular weight of the pelB signal leader sequence conjugated toBrazzein is 8.5 kD. However, after cleavage of the signal peptide, themolecular weight of mature Brazzein is 6.3 kD. The major bandcorresponding to expression of mature brazzein is indicated by arrow inFIG. 4.

In another preferred embodiment, the present invention provides aprocess for extracellular secretion of recombinant Brazzein withimproved yield comprising:

-   -   a) ligating a pET vector with a nucleotide sequence encoding an        ompA signal leader sequence conjugated to brazzein to obtain        recombinant DNA construct and inserting the said construct in        BL21(DE3) E. coli cells by transformation;    -   b) culturing transformed cells of step (a) carrying the        recombinant construct in a culture medium in presence of        Dextrose and inducing protein expression with inducing agents        selected from IPTG or lactose for a period ranging from 2 hrs to        72 hrs, and    -   c) separating culture medium from cells post induction and        purifying Brazzein from the clarified medium.

Accordingly, the process for extracellular synthesis of recombinantbrazzein conjugated to ompA signal leader sequence comprises (a)inserting ompA Brazzein nucleotide coding sequence in a vector, whereinthe said coding sequence is amplified by employing primers selected fromsequences represented by Seq Id No. 4 and Seq Id No. 9; (b) ligatingompA-Brazzein coding sequence in a pET vector to obtain a recombinantconstruct and inserting the said construct in BL21(DE3) E. coli cells bytransformation; (c) culturing the transformed cells of step (b) carryingthe recombinant construct in a culture medium in presence of Dextroseand inducing protein expression with inducing agents selected from IPTGor lactose to obtain secretion of Brazzein into the culture medium, and

(d) separating culture medium from cells post 4-72 hours post inductionand purifying Brazzein from the clarified medium.

In yet another preferred embodiment, the present invention providesBrazzein expressed in the culture medium is selected from wild-typefunctional type III brazzein, a variant or multi-variant of the type IIIform comprising a substitution at the 28^(th) position of the type IIIbrazzein from Aspartate to Alanine D28A.

Accordingly, the present invention provides the production ofextracellular wild-type functional type III Brazzein represented by SeqId No. 8, which is encoded by Seq Id No. 7. Post synthesis amino acids 1to 22 of Seq Id No. 8 representing the pelB signal sequence are cleavedby post translational processes, to yield a mature wild-type functionaltype III Brazzein protein.

In one preferred embodiment, the present invention provides a processfor extracellular synthesis of Brazzein in enhanced yield, wherein theyield of brazzein ranging between 0.5 g/l to 5 g/l.

Accordingly, the present process for extracellular synthesis of Brazzeinis performed in a −1 or 10 litre scale fermenter. Brazzein synthesizedis found to be stable at temperatures ranging from 4° C. to 90° C.

The following examples, which include preferred embodiments, will serveto illustrate the practice of this invention, it being understood thatthe particulars shown are by way of example and for purpose ofillustrative discussion of preferred embodiments of the invention.

EXAMPLES

Source of Expression Host:

E. coli BL21(DE3) cells were commercially obtained from BioBharati LifeSciences, Kolkata, India

Source of Expression Vector:

pET-28a vector was commercially obtained from Novagen.

Example 1: Codon Optimization and Gene Synthesis

The amino acid sequence of the Type III form of Brazzein was retrievedfrom Accession source P56552 (Ming D et al, FEBS Lett.355:106-108(1994)). This amino acid sequence was back translated into anucleotide sequence that was codon optimized for E. coli. The codonoptimized gene also included an Aspartate 28 to an Alanine mutation.This variant was previously shown to exhibit a greater sweet profile incomparison to wildtype Type III Brazzein (Assadi-Porter F M, et al J L.;Arch Biochem Biophys. 2000 Apr. 15; 376(2): 259-65).

The codon optimized gene was fused at the N-terminus with a sequenceencoding for the pelB leader sequence and at the C-terminus with threetandem stop codons. The final codon optimized nucleotide sequence ofpelB-Brazzein is shown in SeqID 1 and the corresponding amino acidtranslation in SeqID 2.

pelB-Brazzein gene was synthesized by Genscript (New Jersey, USA) andcloned into pUC57 cloning vector to generate the plasmid Final PELBRAZ.

Example 2: Construction of pET-pelB-Brazzein

The PCR reaction setup is provided in the Table 1 below:

TABLE 1 Component Concentration Final PELBRAZ plasmid 50 ng primerPelBLun-FNcoNde (Seq Id No. 3) 10 picomoles primer Braz-Rbam (Seq ID No.4) 10 picomoles Pfu-X reaction Buffer 5 μL dNTP mix 1 μM Pfu-Xpolymerase 0.5 μL (1 unit) Sterile water To make up the final volume to50 μL

TABLE 2 Stages of PCR Amplification: Duration Steps Temperature(Minutes) 1 95° C. 5 min 2 95° C. 1 min 3 55° C. 0.5 min 4 72° C. 1 min5 Steps 2, 3 and 4 were — repeated 29 times 6 72° C. 10 mins 7  8° C.hold

The PCR amplification reaction was analyzed on a 1.6% (w/v) Agarose gel.The approximate 250 bp PCR amplification product (FIG. 2) correspondingto pelB-Brazzein was excised from the gel and purified using acommercially available kit. The purified product was digested with NdeIand BamHI for 4 hours at 37° C. and purified using a PCR spin columnkit. This was ligated with pET-28a vector that was previously digestedwith NdeI and BamHI and gel purified. The ligation mixture wastransformed into DH5 alpha competent cells. The transformed cells wereplated out on LB plates containing 50 μg/mL Kanamycin and incubatedovernight at 37° C. Single colonies were picked from the plate into 5 mLLB broth containing 50 ug/mL Kanamycin and grown for 16 hrs in anorbital shaker at 37° C. and 210 rpm. Plasmid DNA was isolated from thecultures and analysed by DNA sequencing. A plasmid clone containing thedesired pelB-Brazzein insert was identified and labelled aspET-pelB-Brazzein and used for protein expression studies.

Example 3: Construction of pET-pelB-Brazzein (A28D)

In order to create wild-type TypeIII Brazzein, amino acid residue number50 of pelB-Brazzein was mutated from an Alanine to an Aspartate and isreferred to as pelB-Brazzein(A28D).

The first PCR reaction was setup with final PELBRAZ plasmid, primerPelBLun-FNcoNde (Seq Id No. 3), primer BRAZ-A28DRSOE (Seq Id No. 5),Pfu-X reaction buffer, dNTP mix, Pfu-X polymerase and sterile water wasused to make up the final PCR reaction solution volume to 50 μL. Thespecific concentrations of the said components are provided in Table 3.

Seq Id No. 3: 5′-GCGCGCCCATGGCATATGAAATACCTGCTGCCGACCGC-3′ Seq Id No. 5:5′-CACCGCTACGCGCATGTTTATCCAGTTTACAGTCGTAGTTACATTG GTTCGC-3′

TABLE 3 PCR solution for the first reaction Components ConcentrationFinal PELBRAZ plasmid 50 ng PelBLun-FNcoNde (Seq Id No. 3) 10 picomolesBRAZ-A28DRSOE (Seq Id No. 5) 10 picomoles Pfu-X reaction Buffer 5 μLdNTP mix 1 μM Pfu-X polymerase 0.5 μL (1 unit) Sterile water To make upthe final volume to 50 μL

TABLE 4 Reaction Conditions Stages of PCR Amplification: Duration StepsTemperature (Minutes) 1 95° C. 5 min 2 95° C. 1 min 3 55° C. 0.5 min 472° C. 1 min 5 Steps 2, 3 and 4 were — repeated 29 times 6 72° C. 10mins 7  8° C. hold

PCR amplification reaction was analyzed on a 1.6% (w/v) Agarose gel. Theapprox. 170 bp PCR amplification product was excised from the gel andpurified using a commercially available gel extraction kit. Thispurified fragment was labelled as Frag 1.

The second PCR reaction was setup with: Final PELBRAZ plasmid, primerBRAZ-A28DFSOE (Seq Id No. 6), primer Braz-Rbam (Seq Id No. 4), Pfu-Xreaction buffer, dNTP mix, Pfu-X polymerase and sterile water was usedto make up the final volume of the second reaction solution to 50 uL.

Seq Id No. 6: 5′-GATAAACATGCGCGTAGCGGTG-3′ Seq Id No. 4:5′-GCGCGCGGATCCTCATTATTAATATTCACAGTAGTCAC AGATACATTGCAG-3′

TABLE 5 PCR solution for the second reaction Components ConcentrationFinal PELBRAZ plasmid 50 ng BRAZ-A28DFSOE (Seq Id No. 6), 10 picomolesBraz-Rbam (Seq Id No. 4), 10 picomoles Pfu-X reaction Buffer 5 μL dNTPmix 1 μM Pfu-X polymerase 0.5 μL (1 unit) Sterile water To make up thefinal volume to 50 μL

PCR amplification was performed by employing the reaction conditions andPCR parameters specified in Table 4. The PCR amplification reaction wasanalyzed on a 1.6% (w/v) agarose gel. The eighty base pair PCRamplification product was excised from the agarose gel and purifiedusing a commercially available gel extraction kit. The final purifiedfragment was labelled as Frag 2.

The third PCR reaction comprised splicing by overlap extension PCRreaction, the said reaction setup comprising Frag1, Frag2, primerPelBLun-FNcoNde (Seq Id No. 3), primer Braz-Rbam (Seq Id No. 4), Pfu-Xreaction Buffer, dNTP mix, Pfu-X polymerase and sterile water was usedto make up the final volume to 50 μL.

TABLE 6 PCR solution for the third reaction Component ConcentrationFrag1 5 μL Frag2 5 μL primer PelBLun-FNcoNde (Seq Id No. 3) 10 picomolesprimer Braz-Rbam (Seq ID No. 4) 10 picomoles Pfu-X reaction Buffer 5 μLdNTP mix 1 μM Pfu-X polymerase 0.5 μL (1 unit) Sterile water To make upthe final volume to 50 μL

PCR amplification was performed by employing the reaction conditions andPCR parameters specified in Table 4.

The PCR amplification reaction was analyzed on a 1.6% (w/v) Agarose gel.The approximate 250 bp PCR amplification product corresponding topelB-Brazzein(A28D) was excised from the gel and purified using acommercially available kit. The purified product was digested with NdeIand BamHI for 4 hours at 37° C. and purified using a PCR spin columnkit. This was ligated with pET-28a vector that was previously digestedwith NdeI and BamHI and gel purified. The ligation mixture wastransformed into DH5 alpha competent cells. The transformed cells wereplated out on LB plates containing 50 μg/mL Kanamycin and incubatedovernight at 37° C. Single colonies were picked from the plate into 5 mLLB broth containing 50 μg/mL Kanamycin and grown for 16 hrs in anorbital shaker at 37° C. and 210 rpm. Plasmid DNA was isolated from thecultures and analysed by DNA sequencing. A plasmid clone containing thedesired pelB-Brazzein(A28D) insert was identified and labelled aspET-pelB-Brazzein(A28D) and used for protein expression studies.

The final nucleotide sequence of pelB-Brazzein(A28D) is shown in Seq IdNo. 7 and the corresponding amino acid translation in Seq Id No. 8.

Example 4: Construction of pET-ompA-Brazzein(A28D)

The ompA-Brazzein(A28D) nucleotide sequence was PCR amplified usingfollowing primers: The PCR reaction was setup withpET-pelB-Brazzein(A28D) plasmid, primer ompANde, primer Braz-Rbam, Pfu-Xreaction buffer, dNTP mix, Pfu-X polymerase and sterile water was usedto make up the final volume to 50 μL. ompANde (Seq Id No. 9):

5′GCGCGCCCATGGCAATGAAAAAAACGGCAATTGCGATAGCGGTTGCGCTAGCTGGTTTTGCCACGGTGGCGCAGGCTGACAAATGTAAAAAGG- 3′

Braz-Rbam (Seq Id No. 4):

5′GCGCGCGGATCCTCATTATTAATATTCACAGTAGTCACAGATACAT TGCAG-3′

The PCR reaction was setup with pET-pelB-Brazzein(A28D) plasmid, primerompANde, primer Braz-Rbam, Pfu-X reaction buffer, dNTP mix, Pfu-Xpolymerase and sterile water was used to make up the final volume to 50μL.

TABLE 5 PCR solution Component Concentration pET-pelB-Brazzein(A28D)plasmid 50 ng ompANde (Seq Id No. 9) 10 picomoles Braz-Rbam (Seq Id No.4) 10 picomoles Pfu-X reaction Buffer 5 μL dNTP mix 1 μM Pfu-Xpolymerase 0.5 μL (1 unit) Sterile water To make up the final volume to50 μL

PCR amplification was performed by employing the reaction conditions andPCR parameters specified in Table 2.

PCR amplification reaction was analyzed on a 1.6% (w/v) agarose gel. Theapprox. 250 bp PCR amplification product corresponding toompA-Brazzein(A28D) was excised from the gel and purified using acommercially available gel extraction kit. The purified product wasdigested with NdeI and BamHI for 4 hours at 37° C. and purified using aPCR spin column kit. This was ligated with pET-28a vector that waspreviously digested with NdeI and BamHI and gel purified. The ligationmixture was transformed into DH5alpha competent cells. The transformedcells were plated out on LB plates containing 50 μg/mL Kanamycin andincubated overnight at 37° C. Single colonies were picked from the plateinto 5 mL LB containing 50 ug/mL Kanamycin and grown for 16 hrs in anorbital shaker at 37° C. and 210 rpm. Plasmid DNA was isolated from thecultures and submitted for DNA sequencing. A plasmid clone containingthe desired ompA-BRAZ insert was identified and labeled aspET-ompA-Brazzein(A28D) and used for protein expression studies.

The final nucleotide sequence of ompA-Brazzein is shown in Seq Id No. 10and the corresponding protein expressed in Seq Id No. 11.

Example 5: Protein Expression

(i) Expression of pelB-Brazzein with IPTG in LB Medium

The pET-pelB-Brazzein plasmid was transformed into BL21(DE3) cells. Thetransformed cells were plated out on LB Agar plates containing 50 μg/mLKanamycin and incubated at 30° C. overnight. A single colony was pickedfrom the plate in 5 mL LB containing 50 μg/mL Kanamycin and 1% (w/v)Dextrose and grown for 16 hours at 30° C. and 180 rpm. The culture wasdiluted into two 250 mL baffled flasks containing 25 mL of LBsupplemented with 50 μg/mL kanamycin, 0.1% dextrose and growth wascontinued at 30° C. and 210 rpm. When optical density (0D600) of thecultures reached 0.4, protein expression was induced by adding IPTG to afinal concentration of 0.25 mM and 0.5 mM and growth continued 30° C.and 210 rpm. Samples were harvested at 24, 48 and 72 hrs post-inductionby spinning down 100 μL of culture and carefully separating thesupernatant from the cell pellet. The cell pellets were stored at −20°C. and the supernatants at 4° C. till further analysis by SDS-PAGE (seeFIGS. 3 and 4).

(ii) Expression of pelB-Brazzein with Lactose in LB medium:

pET-pelB-Brazzein plasmid was transformed into BL21(DE3) cells. Thetransformed cells were plated out on LB Agar plates containing 50 μg/mLKanamycin and incubated at 30° C. overnight. A single colony was pickedfrom the plate in 5 mL LB containing 50 μg/mL kanamycin and 1% (w/v)Dextrose and grown for 16 hours at 30° C. and 180 rpm. The culture wasdiluted into two 250 mL baffled flasks containing 25 mL of LBsupplemented with 50 ug/mL kanamycin and growth was continued at 30° C.and 210 rpm. When optical density (0D600) of the cultures reached 0.4,protein expression was induced by adding Lactose to a finalconcentration of 2.5 mM and 5 mM and growth was continued 30° C. and 210rpm. Samples were harvested at 24, 48 and 72 hrs post-induction byspinning down 100 μL of culture and carefully separating the supernatantfrom the cell pellet. The cell pellets were stored at −20° C. and thesupernatants at 4° C. till further analysis by SDS-PAGE (see FIG. 5).

(iii) Expression of pelB-Brazzein with Lactose in TB Medium:

pET-pelB-Brazzein plasmid was transformed into BL21(DE3) cells. Thetransformed cells were plated out on LB Agar plates containing 50 μg/mLKanamycin and incubated at 30° C. overnight. A single colony was pickedfrom the plate in 5 mL LB containing 50 ug/mL kanamycin and 1% (w/v)Dextrose and grown for 16 hours at 30° C. and 180 rpm. The culture wasdiluted into a baffled flask containing 25 mL of TB supplemented with 50μg/mL kanamycin, 0.1% (w/v) Dextrose and growth was continued at 30° C.and 210 rpm. When optical density (0D600) of the cultures reached 9.0,protein expression was induced by adding Lactose to a finalconcentration of 5 mM and growth continued at 30° C. and 210 rpm.Samples were harvested at 4, 18, 24, 48 and 72 hrs post-induction byspinning down 100 μL of culture and carefully separating the supernatantfrom the cell pellet. The cell pellets were stored at −20° C. and thesupernatants at 4° C. till further analysis by SDS-PAGE (see FIG. 6).

(iv) Expression of pelB-Brazzein(A28D) with Lactose in TB Medium:

pET-pelB-Brazzein(A28D) plasmid was transformed into BL21(DE3) cells.The transformed cells were plated out on LB Agar plates containing 50μg/mL Kanamycin and incubated at 30° C. overnight. A single colony waspicked from the plate in 5 mL LB containing 50 ug/mL kanamycin and 1%(w/v) Dextrose and grown for 16 hours at 30° C. and 180 rpm. The culturewas diluted into a baffled flask containing 25 mL of TB supplementedwith 50 μg/mL kanamycin, 0.1% (w/v) Dextrose and growth was continued at30° C. and 210 rpm. When optical density (0D600) of the cultures reached9.0, protein expression was induced by adding Lactose to a finalconcentration of 5 mM and growth continued 30° C. and 210 rpm. Sampleswere harvested at 4, 18, 24, 48 and 72 hrs post-induction by spinningdown 100 μL of culture and carefully separating the supernatant from thecell pellet. The cell pellets were stored at −20° C. and thesupernatants at 4° C. till further analysis by SDS-PAGE (see FIG. 7).

(v) Expression of ompA-Brazzein(A28D) with Lactose in TB Medium:

pET-ompA-Brazzein(A28D) plasmid was transformed into BL21(DE3) cells.The transformed cells were plated out on LB Agar plates containing 50μg/mL Kanamycin and incubated at 30° C. overnight. A single colony waspicked from the plate in 5 mL LB containing 50 μg/mL kanamycin and 1%(w/v) Dextrose and grown for 16 hours at 30° C. and 180 rpm. The culturewas diluted into a baffled flask containing 25 mL of TB supplementedwith 50 μg/mL kanamycin, 0.1% (w/v) Dextrose and growth was continued at30° C. and 210 rpm. When optical density (0D600) of the cultures reached9.0, protein expression was induced by adding Lactose to a finalconcentration of 5 mM and growth continued 30° C. and 210 rpm. Sampleswere harvested at 4, 18, 24, 48 and 72 hrs post-induction by spinningdown 100 μL of culture and carefully separating the supernatant from thecell pellet. The cell pellets were stored at −20° C. and thesupernatants at 4° C. till further analysis by SDS-PAGE (see FIG. 8).

(vi) Expression of pelB-Brazzein(A28D) in Fermentor by Fed-BatchCultivation

Fermentor based expression of pelB-Brazzein(A28D) was carried out in a 5l fermentor by fed-batch cultivation. pET-pelB-Brazzein(A28D) plasmidwas transformed in E. coli BL21(DE3) cells. The transformed cells wereplated out on LB Agar plates containing 50 μg/mL Kanamycin and incubatedat 30° C. overnight. A single colony was picked from the plate in 5 mLLB containing 50 μg/mL kanamycin and 1% (w/v) Dextrose and grown for 16hours at 30° C. and 180 rpm. A 1.5 mL aliquot of the overnight culturewas diluted into a baffled flask containing 150 mL of LB supplementedwith 50 μg/mL kanamycin, 1% (w/v) Dextrose and growth was continued for12 hours at 30° C. and 210 rpm. 4.2 L of sterile TB medium in thefermentor were inoculated with 100 ml of this culture and supplementedwith 0.1% (w/v) Dextrose and 50 μg/mL Kanamycin. During thefermentation, the temperature and pH were maintained at 30° C. and 7.0,respectively. The dissolved oxygen level was maintained at 30-40% byusing air or pure oxygen and the speed was maintained at 600 rpm. Afterthe optical density (0D600) reached 10, a final concentration of 5 mMLactose was added to induce the expression of pelB-Brazzein(A28D).Samples of the culture were harvested at 4, 12, 18 and 24 hours postinduction by spinning down 5 mL of culture and carefully separating thesupernatant from the cell pellet. The cell pellets were stored at −20°C. and the supernatants at 4° C. till further analysis by SDS-PAGE (seeFIG. 9).

(vii) Purification of Brazzein(A28D)

The culture from the fermentor was spun at 5000 g and the clarified cellfree supernatant was transferred to a fresh container. To this, powdered1.2 kg ammonium sulfate was slowly added with gentle stirring. Themixture was incubated for 1 hour with gentle stirring at roomtemperature and subsequently spun at 5000 g for 30 mins. The supernatantobtained after the spin was discarded and pellet containing precipitatedbrazzein and other proteins was dissolved in 200 mL of deionized water.This solution was heated to 90° C. for 1 hour and subsequently spun at5000 g for 30 mins. The supernatant was carefully decanted andtransferred to a fresh vessel and it was found that Brazzein (A28D)constitutes a major fraction of the total protein in this supernatant.The supernatant was passed through a 10 kDa MW cut-off centrifugalconcentrator at 3000 g. Majority Brazzein passes through the filtermembrane and is collected in the flow-through fraction while remaininghigher molecular weight proteins do not pass through the membrane andare retained as the retentate fraction in the concentrator. Theflow-through fraction was transferred to a 2 kDa MW cut-off centrifugalconcentrator and buffer exchanged to water by repeated concentration anddilution with deionized water. This was continued till the colour of theretentate fraction in the concentrator was colourless and theflow-through fraction did not have any salty taste. During this, it wasfound that Brazzein was completely retained by the membrane in theretentate fraction while low molecular weight molecules passed throughinto the flow-through fraction (see FIG. 10).

The retentate fraction containing Brazzein was harvested into a freshvessel and stored at 4° C. for further analysis. Protein Quantitation ofthe sample gave a final yield of 4.4 grams of Brazzein per litre ofculture. Purity of Brazzein in lane 2 of FIG. 10 is greater than 95%pure by SDS-PAGE analysis.

(viii) Sensory Analysis of Brazzein

A portion of the purified Brazzein(A28D) was lyophilized andre-dissolved in deionized water to 1.0 mg/mL. From this, Brazzein(A28D)solutions with following concentrations were made: 0.5, 1.0, 2.0, 3.0,4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 μg/mL. A 1% (w/v) sucrosesolution, the lowest concentration of sucrose detectable by humans, wasused as a reference. The taste panel consisted of fifteen females andfifteen males with normal health and normal sense of taste.Two-hundred-microliter samples were applied to the anterior region ofthe tongue. After each test, the mouth was rinsed with tap water. Thetasters sampled from the lower concentration samples to higher. Eachtaster chose the first sample that could be sensed for sweetness.Sweetness potencies were reported relative to sucrose on a weight basis.The purified Brazzein(A28D) was found to be 1660 times sweeter thansucrose on a weight basis.

TABLE 6 Sensory Analysis of Purified Brazzzein Experimental ThresholdRelative Sweetness Molecule % [g/100 mL] (by weight) Sucrose 1 1Brazzein(A28D) 0.0006 1660

(ix) SDS-PAGE Analysis

Protein expression was analyzed by SDS-PAGE on 15% Tris-Glycine gels. Toanalyze protein in the cell culture medium, 17 μL of cell freesupernatant (corresponding to 0.07% of total culture volume) was mixedwith 17 μL of 2× reducing sample buffer, heated for 5 mins in a PCRmachine, briefly spun and loaded into the gel. The gel was run at aconstant voltage of 125V till the dye front exited the gel. The gelswere washed in Milli-Q water for 1 hour and stained with CoomassieStain. To analyze protein in whole cells, frozen cell pellets werethawed, re-suspended in 50 μL of Milli-Q water. 17 μL of resuspendedcells were mixed with 17 μL of 2× Reducing Sample Buffer, heated for 5mins in a PCR machine, briefly spun and loaded into the gel. The gel wasrun at constant voltage of 125V till the dye front exited it. The gelswere washed in Milli-Q water for 1 hour and stained with CoomassieStain.

(x) Thermal Stability and Protein Estimation

Cell free supernatant from (i) was heated to 90° C. for 1 hour in awater bath and spun at 17,500 g for 30 mins. The supernatant wasanalyzed by SDS-PAGE. Protein quantitation was done using BCA assay kit.As a result of heating, most of the endogenous E. coli proteins presentin the cell free supernatant precipitated, leaving >90% pure Brazzein insolution. Protein estimation of the supernatant from the heated and spunsample demonstrated a yield of 0.56 g/L of Brazzein (FIG. 11).

(xi) N-Terminal Sequencing

Protein was separated on SDS-PAGE and transferred onto a 0.22 urn poresize PVDF membrane. The membrane was stained with Coommassie Blue tillprotein bands appeared. The membrane was transferred to Milli-Q water.The band corresponding to Brazzein was cut out with a clean scalpel andsubmitted for N-terminal sequencing of the first five amino acids.Results from the N-terminal sequencing matched the expected sequence ofType III Brazzein.

1-10. (canceled)
 11. A culture medium for manufacturing a Brazzeinpeptide, the culture medium comprising: a nutrient broth; and an E. colicell, the E. coli cell comprising a recombinant nucleic acid, whereinthe recombinant nucleic acid comprises: (i) a nucleic acid sequenceencoding the Brazzein peptide, (ii) a nucleic acid sequence encoding asignal leader amino acid sequence, wherein the signal leader amino acidsequence is fused at a 5′ end of the Brazzein peptide, and wherein thesignal leader amino acid sequence is operable to direct extracellularsecretion of the Brazzein peptide, and (iii) an expression controlsequence operable to direct expression of the nucleic acid sequenceencoding the signal leader amino acid sequence.
 12. The culture mediumof claim 11, wherein the Brazzein peptide has an amino acid sequenceselected from the group consisting of: amino acids 23-75 of SEQ ID NO: 8and amino acids 23-75 of SEQ ID NO:
 2. 13. The culture medium of claim11 further comprising an inducing agent, wherein the expression controlsequence is lactose inducible, and wherein the inducing agent isselected from the group consisting of: Lactose, isopropylthiogalactoside(IPTG), and a Lactose analogue.
 14. The culture medium of claim 11,further comprising the Brazzein peptide, wherein the Brazzein peptidehas a concentration of at least 0.5 g/L.
 15. The culture medium of claim11, further comprising the Brazzein peptide, wherein the Brazzeinpeptide has a concentration of at least 4.4 g/L.
 16. A process ofsecreting a Brazzein peptide into a nutrient broth, the processcomprising: culturing an E. coli cell comprising a recombinant nucleicacid in the nutrient broth to generate an E. coli culture, wherein therecombinant nucleic acid comprises: (i) a nucleic acid sequence encodingthe Brazzein peptide, (ii) a nucleic acid sequence encoding a signalleader amino acid sequence, wherein the signal leader amino acidsequence is fused at a 5′ end of the Brazzein peptide, and wherein thesignal leader amino acid sequence is operable to direct extracellularsecretion of the Brazzein peptide, and (iii) an expression controlsequence operable to direct expression of the nucleic acid sequenceencoding the signal leader amino acid sequence; and activating theexpression control sequence.
 17. The process of claim 16, the activatingthe expression control sequence further comprising adding an inducingagent to the nutrient broth.
 18. The process of claim 17, wherein theexpression control sequence is lactose inducible, and wherein theinducing agent is selected from the group consisting of: Lactose,isopropylthiogalactoside (IPTG), and a Lactose analogue.
 19. The processaccording to claim 16, further comprising: periodically measuring anoptical density (OD 600) of the E. coli culture, and adding an inducingagent to the nutrient broth when the E. coli culture has an opticaldensity (OD 600) of 0.4.
 20. The process according to claim 16, whereinthe Brazzein peptide has an amino acid sequence selected from the groupconsisting of: amino acids 23-75 of SEQ ID NO: 8 and amino acids 23-75of SEQ ID NO:
 2. 21. The process according to claim 16, wherein thesignal leader sequence is selected from the group consisting of pelB,ompA, Bla, PhoS, MalE, LivK, LivJ, MglB, AraF, AmpC, RbsB, MerP, CpdB,Lpp, LamB, OmpC, PhoE, OmpF, TolC, BtuB, and LutA.
 22. A process forisolating a Brazzein peptide from a nutrient broth, the processcomprising: culturing an E. coli cell comprising a recombinant nucleicacid in the nutrient broth to generate an E. coli culture, wherein therecombinant nucleic acid comprises: (i) a nucleic acid sequence encodinga Brazzein peptide, (ii) a nucleic acid sequence encoding a signalleader amino acid sequence fused to a 5′ end of the nucleic acidsequence encoding the Brazzein peptide, wherein cellular translationgenerates the Brazzein peptide having the signal leader amino acidsequence located at a 5′ end, wherein the signal leader amino acidsequence is operable to direct extracellular secretion of the Brazzeinpeptide, and (iii) an expression control sequence operable to directexpression of the nucleic acid sequence encoding the signal leader aminoacid sequence; activating the expression control sequence; andseparating the E. coli culture to form a cell pellet and a supernatant,wherein the supernatant comprises the Brazzein peptide.
 23. The processaccording to claim 22 further comprising purifying the supernatant toyield a purified Brazzein peptide.
 24. The process of claim 22, theactivating the expression control sequence further comprising adding aninducing agent to the nutrient broth.
 25. The process according to claim22, wherein the Brazzein peptide has an amino acid sequence selectedfrom the group consisting of: amino acids 23-75 of SEQ ID NO: 8 andamino acids 23-75 of SEQ ID NO: 2.